A Highly Sensitive Tissue-specific qRT-PCR-based Assay for Detection of Melanoma Cells in Tumor-Draining Lymph Nodes

Melanoma invasion of regional lymph nodes, a critical event in the progression of this disease, is well documented as a poor prognostic factor for patients with melanoma. Assessing lymph node involvement is therefore a routine part of the diagnostic workup for patients presenting with melanoma at a T stage of ≥T2a. In clinical settings, the status and degree of melanoma lymph node involvement is traditionally characterized by histopathological analysis of tissue obtained during a sentinel lymph node biopsy, a labor-intensive and costly approach that requires technically challenging sample preparation and interpretation by a trained pathologist. Alternative approaches that might reduce the financial burden and turnaround time of a sentinel lymph node biopsy workup are therefore desirable. Likewise, the ability to accurately assess lymph node invasion by melanoma in experimental settings is necessary to gain new insights into mechanisms of distant metastasis and tumor-associated immune suppression. With these applications in mind, we recently developed, and describe herein, a tissue-specific qRT-PCR-based protocol for detecting melanoma cells within tumor-draining lymph nodes. Using murine models of lymph-node-invasive and lymph-node-noninvasive melanoma cell lines and a melanin-biosynthesis-pathway-specific Trp2 gene expression assay, we validated this method as a highly sensitive strategy for assessing lymph node involvement by melanoma. © 2025 Wiley Periodicals LLC. Basic Protocol 1: Growth and maintenance of murine melanoma cell cultures Basic Protocol 2: Extraction and quantification of RNA from murine melanoma cultures Basic Protocol 3: cDNA synthesis via reverse transcription Basic Protocol 4: Probe and primer design for TaqMan-based qPCR Basic Protocol 5: qPCR analysis of tissue-specific Trp2 gene expression Basic Protocol 6: In vitro validation of qRT-PCR Trp2 gene expression assay for detection of melanoma cells in murine whole lymph node preparations Basic Protocol 7: In vivo validation of qRT-PCR Trp2 gene expression assay for detection of melanoma cells in murine tumor-draining lymph nodes.